Background and purpose: GABAA receptors containing δ-subunits are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA-evoked current amplitudes. Thus, there are some published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of δ-containing receptors. The aim of this study was to obtain robust homogenous expression of α1βδ receptors for in-depth investigation.
Experimental approach: Novel δ-containing pentameric concatenated constructs were designed. The resulting α1β2δ and α1β3δ GABAA receptor concatemers were investigated by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes.
Key results: First, while homogenous α1βδ GABAA receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free α1, β2/3, and δ subunits, concatenated pentameric α1β2δ and α1β3δ constructs resulted in robust expression levels of concatemers. Second, by using optimised constructs that give unidirectional assembly of concatemers, we found that the δ subunit cannot directly participate in GABA binding and receptor activation. Hence, functional δ-containing receptors are likely to all have a conventional 2α:2β:1δ stoichiometry arranged as βαβαδ when viewed counterclockwise from the extracellular side. Third, α1β2/3δ receptors were found to express efficiently in X. laevis oocytes but have a low estimated open probability of ~0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by, for example, neurosteroids.
Conclusion and implications: Our data answer important outstanding questions regarding the pharmacology and stoichiometry of α1δ-containing GABAA receptors and pave the way for future analysis and drug discovery efforts.
Keywords: DS2; GABA; allopregnanolone; ethanol; etomidate; receptor; stoichiometry.
© 2021 The British Pharmacological Society.